Cystatin M is a potent endogenous inhibitor of lysosomal cysteine proteases. In breast carcinoma, cystatin M expression is frequently downregulated. It has been shown that cystatin M expression suppressed growth and migration of breast cancer cells. We examined the methylation status of the CpG island promoter of cystatin M in four breast cancer cell lines (MDAMB231, ZR75-1, MCF7 and T47D), in 40 primary breast carcinoma and in corresponding normal tissue probes by combined bisulphite restriction analysis. To investigate the effects of cystatin M expression on the growth of breast carcinoma, cystatin M was transfected in T47D. The cystatin M promoter was highly methylated in all four-breast cancer cell lines. Primary breast tumours were significantly more frequently methylated compared to normal tissue samples (60 vs 25%; P=0.006 Fisher's exact test). Treatment of breast cancer cells with 5-aza-2′-deoxycytidine (5-Aza-CdR), reactivated the transcription of cystatin M. Transfection of breast carcinoma cells with cystatin M caused a 30% decrease in colony formation compared to control transfection (P=0.002). Our results show that cystatin M is frequently epigenetically inactivated during breast carcinogenesis and cystatin M expression suppresses the growth of breast carcinoma. These data suggest that cystatin M may encode a novel epigenetically inactivated candidate tumour suppressor gene.
Cysteine proteases are known to play a crucial role in cancer diseases (Krepela, 2001). Cysteine proteases can be involved in enhanced cell survival and proliferation, escape from immune surveillance, cell adhesion and migration, remodelling and invasion of the extracellular matrix (Nomura and Katunuma, 2005). Cystatins are potent inhibitors of endogenous mammalian lysosomal cysteine proteases and protect cells against uncontrolled proteolysis (Abrahamson et al., 2003). Disturbance of the balance between proteases and their cystatins can lead to irreversible damage and tumour development (Calkins and Sloane, 1995; Henskens et al., 1996). Cystatin M is a new member of the human cystatin gene family and demonstrates more diverse tissue distribution and target specificity than other cystatins (Ni et al., 1997; Sotiropoulou et al., 1997). Cystatin M was first identified and cloned by differential RNA display as a transcript that is downregulated in metastatic breast cancer cells when compared to primary breast cancer cells (Ni et al., 1997; Sotiropoulou et al., 1997). Cystatin M is a low molecular mass protein sharing 27–32% homology to other cystatins. The protein can be secreted from cells in a glycosylated (17 kDa) and unglycosylated (14.4 kDa) form (Ni et al., 1997). It has been reported that constitutive expression of cystatin M in human breast cancer cells MDA-MB-435S significantly reduces in vitro cell proliferation, migration, Matrigel invasion and tumour–endothelial cell adhesion (Shridhar et al., 2004). Moreover, the ectopic expression of cystatin M in scid mice strongly delayed tumour growth and lowered the rate of metastases in lung and liver (Zhang et al., 2004).
Cystatin M has been assigned to chromosome region 11q13, which is the site of loss of heterozygosity (LOH) in several cancer types and is believed to harbour tumour suppressor genes (Stenman et al., 1997; Srivatsan et al., 2002). Cystatin M regulates the activity of cathepsin B and cathepsin L, which are the two most important cysteine proteases implicated in tumour cell invasion and metastasis (Lah and Kos, 1998; Frosch et al., 1999). Cystatin M is consistently expressed in normal and premalignant breast epithelial cells, but downregulated in malignant breast cancer cell lines (Sotiropoulou et al., 1997). However the mechanisms of cystatin M downregulation has not been elucidated.
Epigenetic inactivation of tumour suppressor genes plays a fundamental role in the development and progression of cancer (Jones and Baylin, 2002). To determine if epigenetic silencing of cystatin M occurs in pathogenesis of breast cancer, we investigated the methylation status of the cystatin M promoter in primary breast carcinoma. Moreover, we transfected cystatin M into breast cancer cells and analysed colony formation.
Hypermethylation of the cystatin M CpG island in breast cancer
It has been reported that cystatin M expression is significantly reduced in breast tumours (Sotiropoulou et al., 1997) and that its expression inhibits growth of breast cancer cells (Shridhar et al., 2004). Epigenetic inactivation of tumour suppressor genes by hypermethylation of their CpG island promoters plays an essential role in tumour pathogenesis (Herman and Baylin, 2003). Therefore, we investigated whether aberrant methylation of the cystatin M promoter occurred in breast cancer. The promoter region of cystatin M was screened for the presence of CpG dinucleotides by in silicio analysis (Figure 1a). A 500 bp long CpG island covering the transcription initiation site and the first exon was revealed. To elucidate methylation of cystatin M promoter in breast cancer, we performed combined bisulphite restriction analysis (COBRA) assays (Xiong and Laird, 1997). First, we analysed the methylation status of the cystatin M promoter in four breast carcinoma cell lines (ZR75-1, MCF7, T47D and MDAMB231; Figure 1b). ZR75-1 and MCF7 were completely methylated and MDAMB231 and T47D were partially methylated (Figure 1b). We confirm the methylation status of the cystatin M promoter by sequencing polymerase chain reaction (PCR) products obtained from breast cancer cell lines ZR75-1 and T47D, and from a normal breast tissue sample (Figure 2). Subsequently, we analysed methylation of the cystatin M CpG island promoter in primary breast carcinomas and corresponding normal tissue samples (Figure 1c). In 24 out of 40 (60%) breast carcinomas, the promoter of cystatin M was methylated. In contrast, in only seven out of 28 (25%) normal breast tissue samples cystatin M was methylated (Table 1). However, methylation was lower than in the corresponding tumour cases. Hypermethylation was significantly more frequent in tumours compared to normal samples (P=0.006, Fisher's exact test). Methylation of cystatin M was compared with different clinicopathological data. No significant correlation with tumour entities, grading and age of onset was found (Table 1). It was observed that oestrogen receptor-positive tumours were more frequently methylated than oestrogen receptor-negative tumours. This tendency was not significant for cystatin M (P=0.061), but was significant for Ras association domain family 1A (RASSF1A) (P=0.009).
The methylation status of the RASSF1A tumour suppressor gene for the analysed primary breast cancer samples was available from the previous work (Dammann et al., 2001) (Table 1). We observed a significant association between methylation of cystatin M and RASSF1A. Primary breast cancer cases with methylated cystatin M had a significantly increased frequency of RASSF1A methylation (79%) compared to cases with unmethylated cystatin M (38%, P=0.018 Fisher's exact test).
Inhibition of DNA methylation reactivates cystatin M expression
To correlate the methylation status with cystatin M transcription, we performed reverse trancriptase–polymerase chain reaction (RT–PCR) in breast cancer cell lines T47D and ZR75-1, and in normal human mammary epithelial cells (HMEC) (Figure 3). High level of cystatin M transcript was detected in HMEC. In T47D, which harbours a partially methylated promoter, very low level of cystatin M was found, and in the completely methylated ZR75-1 cells, no cystatin M was observed (Figure 3). Subsequently, we treated these cancer cell lines with two concentrations (5 and 10 μ M) of 5-Aza-CdR, a drug that inhibits DNA methylation (Jones and Taylor, 1980). After 4 days of treatment, we detected an increase of cystatin M transcripts in breast cancer cell lines T47D and ZR75-1 (Figure 3).
Transfection of cystatin M inhibits colony formation
Previously, it has been shown that cystatin M expression inhibits growth of the breast cancer cell line MDA-MB-435SD (Shridhar et al., 2004). To confirm function of cystatin M as a tumour suppressor gene in breast carcinogenesis, we transfected the breast cancer cell line T47D with cystatin M and with a vector control (Figure 4). After selection with G418 for 4 weeks, we observed a 30% decrease in colony formation in cells transfected with cystatin M (n=46±3) in comparison to T47D cells transfected with a vector control (n=65±4) (P=0.002; one-way analysis of variance (ANOVA) test).
Cystatin M, a potent inhibitor of human lysosomal proteases, was identified as a gene frequently transcriptionally downregulated during progression of breast cancer (Ni et al., 1997; Sotiropoulou et al., 1997; Zhang et al., 2004). In our study, we demonstrated that hypermethylation of the cystatin M promoter occurs frequently in primary breast carcinomas and breast cancer cell lines. This result suggests that aberrant methylation of the cystatin M CpG island is an important mechanism of its silencing. To our knowledge, this is the first demonstration of epigenetic silencing of cystatin M in cancer. Complete loss of expression of cystatin M was previously demonstrated in different metastatic tumour cell lines (Sotiropoulou et al., 1997). Here we show that in several breast cancer cell lines cystatin M is highly reduced and was re-expressed after inhibition of DNA methyltransferase with 5-Aza-CdR. These data confirm that hypermethylation of the cystatin M promoter inhibits its transcription. It has been suggested that cystatin M inactivation is associated with progression of a primary tumour to a metastatic phenotype (Sotiropoulou et al., 1997). Subsequently, it has been demonstrated that constitutive expression of cystatin M in human breast carcinoma MDA-MB-435S cells reduces in vitro cell proliferation, migration, Matrigel invasion and adhesion to endothelial cells (Shridhar et al., 2004). Our data support this result as re-expression of cystatin M in the breast cancer cells T47D significantly reduced colony formation of these cells. In vivo experiments with scid mice transplanted with breast cancer cells expressing cystatin M showed significantly delayed breast tumour growth and lowered metastatic burden in the lung and liver at secondary sites (Zhang et al., 2004). Thus, it has been proposed that cystatin M may function as candidate tumour suppressor for breast carcinogenesis (Zhang et al., 2004).
Cystatin M is a potent inhibitor of cysteine peptidases, including papain, cathepsin B, cathepsin V and cathepsin L (Ni et al., 1997; Cheng et al., 2006). These peptidases play key roles in intracellular protein degradation. Cathepsins B and L promote tumour growth, invasion and metastasis through degradation of extracellular connective matrices and through endothelial cell growth-directed activities (Krepela, 2001). Thus, deregulation of cystatin M in tumours may enhance tumorigenic and metastatic function of cathepsins. Re-expression of cystatin M in breast cancer cells reduced the level of the potent mitogenic and angiogenic factor autotaxin (Song et al., 2006). Additionally, cystatin M has important regulatory functions in human epidermal differentiation (Zeeuwen, 2004). In wound healing, cystatin M expression was not found in the edge of migrating keratinocytes and in epidermal neoplasia cystatin M was only detected in differentiated cells and keratinized cell nests (Zeeuwen et al., 2002). Cystatin M downregulation was also observed in different cancer cells, including prostate cancer, colon cancer, glioblastoma, lung cancer and melanoma (Sotiropoulou et al., 1997; Shridhar et al., 2004). This indicates that cystatin M may be epigenetically downregulated in other cancers. In contrast, in oropharyngeal and skin squamous cell carcinoma, an upregulation of cystatin M expression was reported (Vigneswaran et al., 2003; Haider et al., 2006). Further studies should clarify if aberrant methylation (hyper- or hypomethylation) of cystatin M is a frequent process in pathogenesis of different cancer entities.
In our previous work, we have identified a novel Ras effector homologue termed RASSF1A (Dammann et al., 2000). It has been demonstrated that RASSF1A is frequently downregulated in breast cancer and this silencing was associated with aberrant methylation of the RASSF1A CpG island promoter (Burbee et al., 2001; Dammann et al., 2001). Comparison of methylation of cystatin M with the tumour suppressor gene RASSF1A showed a significant correlation. Previously, we have shown that progressive RASSF1A hypermethylation was found during senescence of normal human mammary epithelial cells (Strunnikova et al., 2005). Methylation of matching normal tissue of breast cancer patients may be related to methylation reported in ageing or attributed to epigenetic field defect and infiltrating tumour cells (Ahuja and Issa, 2000; Waki et al., 2003; Guo et al., 2004).
In summary, our results indicate that cystatin M may function as an epigenetically silenced tumour suppressor in breast carcinogenesis. These results support the suggestion that cystatin M inactivation is a characteristic process in the progression of breast cancer and may play an important role in the safeguarding against this cancer type. The exact role of cystatin M inactivation combined with overexpression of cysteine proteases and the consequential proteolytic imbalance remains to be elucidated.
Materials and methods
Tissues and cell lines
In our study, we analysed 40 primary breast carcinoma and 28 corresponding normal breast tissues. All tissues were classified and obtained from the Pathology Department of the City of Hope National Medical Center (Duarte, CA, USA) and were described previously (Dammann et al., 2001). Four human breast cancer cell lines ZR75-1, T47D, MDAMB231 and MCF7 were obtained from the American Type Culture Collection and were cultured in the recommended growth medium. RNA was isolated and RT–PCR was performed as described below. Genomic DNA was extracted from frozen tissues and cultured cells by a standard phenol/chloroform procedure.
Bisulphite treatment of DNA and methylation analysis
Methylation of the cystatin M promoter region was determined by bisulphite modification of genomic DNA (Clark et al., 1994; Dammann et al., 2000). Combined bisulphite restriction analysis (COBRA) was performed as described previously (Xiong and Laird, 1997). Briefly, 100 ng of bisulphite-treated DNA were amplified with primers CystMU1: 5′-IndexTermTTGTATTGGTATTTGTTGTTGGGGATTG and CystML1: 5′-IndexTermCAAAATACCACCAAAACCAAACCCAAC. A hot-started PCR in 25 μl reaction buffer containing 0.2 mM dNTP mix, 1.5 mM MgCl2, 10 pmol of each primer and Taq polymerase (InViTek GmBH, Berlin, Germany) at 92°C for 30 s, 60°C for 30 s, and 72°C for 30 s for 20 cycles was performed. A seminested PCR was carried out using an internal primer CystMU2: 5′-IndexTermGGGTTAGGTGTGTTTTGGAGGGTAGG and primer CystML1 with similar conditions as described above, but for 30 cycles. Expected product size after seminested PCR was 287 bp. For restriction analysis, PCR products were digested with 10 U of Taq-I (New England Biolabs, Beverly, MA, USA) according to conditions specified by the manufacturer and analysed on a 2% Tris-borate ethylenediamine tetra acetic acid (EDTA) agarose gel.
5-Aza-CdR treatment and RT–PCR analysis
Two breast cancer cell lines (ZR75-1 and T47D) and normal breast epithelial cells (HMEC) were treated with 5-Aza-CdR) (Sigma, Taufkirchen, Germany). Briefly, 2 × 106 cells were grown for 4 days in the presence of different concentrations (0, 5 and 10 μ M) of 5-Aza-CdR. RNA was isolated using Trizol-Reagent (Invitrogen, Karlsruhe, Germany). Reverse transcription was performed with poly-dT-primer and 1 μg RNA in 25 μl of RT-mix with avian myeloblastosis virus-reverse transcriptase (AMV-RT) (Promega GmBH, Heidelberg, Germany) for 1 h at 42°C. Subsequently, 2 μl of cDNA was amplificated at 60°C, for 38 cycles using following cystatin M-specific primers: forward primer, 5′-IndexTermGCAGAAGGCGGCGCAGGC and reverse primer, 5′-IndexTermCTCCTGCTGCGCCCCTGCTG with an expected product size of 223 bp. Amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was carried out to verify integrity of RNA. PCR products were separated on 2% Tris-borate EDTA agarose gels.
Cystatin M transfection and colony formation assay
To transfect cystatin M into the breast cancer cell line T47D, we cloned cystatin M into the mammalian expression vector pcDNA3.1+ vector (Invitrogen, Carlsbad, CA, USA). We obtained plasmid IMAGP998J016038Q from RZPD (Deutsches Ressourcenzentrum für Genomforschung GmbH, Berlin, Germany), which contains the full-length cDNA clone of cystatin M (509 bp). The cDNA of cystatin M was isolated by restriction with HindIII and EcoRI (New England BioLabs GmbH, Frankfurt am Main, Germany) and ligated into pCDNA3.1+ vector. Subsequently, 0.3 μg of plasmid DNA was transfected into T47D cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for 6 h and cells were selected in Rosewell's Park Memorial Institute (RPMI) (Biochrom AG, Berlin, Germany) with 320 μg/ml G418 (PAA Laboratories GmbH, Pasching, Germany). After 4 weeks, clones were stained with Giemsa and colony formation was evaluated. As a negative control, T47D cells transfected with pCDNA3.1+ were utilized. Colony formation assay was repeated three times and means were determined.
Statistical analysis was carried out using SPSS 12. Categorical variables were plotted into contingency tables and evaluated using the Fisher's exact test. Results of colony formation tests were plotted as mean±s.d. Mean values of colony formations were compared using one-way ANOVA test. All reported P-values are two-sided and considered significant when P was <0.05.
combined bisulphite restriction analysis
- RASSF1A :
Ras association domain family 1A
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This study was supported by Grants BMBF FKZ01ZZ0104 and DFG DA552-1 to Reinhard Dammann and WR (FKZ13/13) to Undraga Schagdarsurengin and NIH Grant CA88873 to Gerd P Pfeifer.
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Schagdarsurengin, U., Pfeifer, G. & Dammann, R. Frequent epigenetic inactivation of cystatin M in breast carcinoma. Oncogene 26, 3089–3094 (2007). https://doi.org/10.1038/sj.onc.1210107
- cystatin M
- tumour suppressor gene
- epigenetic inactivation
- breast cancer
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